We study the mechanism and regulation of protein synthesis in eukaryotic cells focusing on regulation by GTP-binding (G) proteins and protein phosphorylation. The first step of protein synthesis is binding the initiator Met-tRNA to the small ribosomal subunit by the factor eIF2. The eIF2 is a GTP-binding protein and during the course of translation initiation the GTP is hydrolyzed to GDP. The eIF2 is released from the ribosome in complex with GDP and requires the guanine-nucleotide exchange factor eIF2B to convert eIF2-GDP to eIF2-GTP. This exchange reaction is regulated by a family of kinases that specifically phosphorylate the alpha subunit of eIF2 on serine at residue 51, and thereby covert eIF2 into an inhibitor of eIF2B. Among the family of eIF2alpha kinases are GCN2 (activated under conditions of amino acid starvation), PKR (activated by double-stranded RNA and downregulates protein synthesis in virally infected cells), and PERK (activated under conditions of ER stress). The factor eIF2 is composed of three polypeptide chains. The gamma subunit of eIF2 is a GTPase that, based on sequence and the structure of the archaeal homolog aIF2gamma, resembles elongation factor EF-Tu. However, in contrast to EF-Tu, which binds tRNAs to the A-site of the 70S ribosome, eIF2 binds Met-tRNAi to the P-site of the 40S subunit. To gain insights into how eIF2 binds Met-tRNAi and then associates with the 40S ribosome, we used directed hydroxyl radical probing to identify eIF2 contacts within the 40SeIF1eIF1AeIF2GTPMet-tRNAimRNA (48S) complex. Based on the structure of the EF-Tu ternary complex, we predicted that linkage of Fe(II)-BABE, a hydroxyl radical generator, to domain III of eIF2gamma would result in cleavage of Met-tRNAi in the T-stem. However, this instead resulted in cleavage of the D-stem of Met-tRNAi and of 18S rRNA at the top of helix h44, a prominent landmark on the intersubunit surface of the 40S subunit. Based on the results of these and other cleavage experiments, and the fact that Met-tRNAi is bound to the P-site of the 40S subunit, we generated a model of the 48S complex in which domain III of eIF2gamma binds near 18S rRNA helix h44 and eIF2gammaMet-tRNAi contacts are restricted to the acceptor stem of the tRNA. In this model of the eIF2 ternary complex, the Met-tRNAi is rotated nearly 180 degrees relative to the position of the tRNA in the EF-Tu ternary complex. Consistent with the alternate models of the eIF2 and EF-Tu ternary complexes, we found that a domain III mutation in EF-Tu severely impaired Phe-tRNA binding; whereas, the corresponding eIF2gamma mutation did not impair Met-tRNAi binding to eIF2. In addition, mutation of conserved positively charged residues on the surface of domain III of eIF2gamma impaired binding of the eIF2GTPMet-tRNAi ternary complex to the 40S ribosome. Thus, despite their structural similarity, eIF2 and EF-Tu bind tRNA in substantially different manners, and we propose that the tRNA-binding domain III of EF-Tu has acquired a new function in eIF2gamma to bind the ribosome. Whereas protein synthesis is known to play a critical role in learning and memory in diverse model systems, human intellectual disability syndromes have not been directly associated with alterations in protein synthesis. Moreover, the consequences of partial loss of eIF2gamma function or eIF2 integrity are unknown in mammals, including humans. Our collaborators in Israel and Germany recently identified a human X-chromosomal neurological disorder characterized by intellectual disability and microcephaly. Mapping studies identified the causative mutation as a single base change in resulting in a missense mutation in eIF2gamma (encoded by EIF2S3). Biochemical studies of human cells overexpressing the eIF2&#947; mutant and of yeast eIF2&#947; with the analogous mutation revealed a defect in binding the eIF2&#946; subunit to eIF2&#947;. Consistent with this loss of eIF2 integrity, the mutation in yeast eIF2&#947; impaired translation start codon selection and eIF2 function in vivo in a manner that was suppressed by overexpression of eIF2&#946;. These findings directly link intellectual disability to impaired translation initiation, and provide a mechanistic basis for the human disease due to partial loss of eIF2 function. Previously, we demonstrated that, when expressed in yeast, human PKR could phosphorylate the alpha subunit of eIF2 on Ser51 resulting in an inhibition of protein synthesis and yeast cell growth. We also identified the mechanism of activation of PKR that requires back-to-back dimerization of two PKR kinase domains. Further demonstrating the importance of kinase domain dimerization, we showed that appending heterologous dimerization domains to the PKR kinase domain activated the kinase. Kinases resembling the eIF2alpha kinases have been identified in the genome sequences of a variety of eukaryotes including pathogens such as Plasmodium falciparum, the protozoan that causes malaria. Plasmodium expresses three kinases related by sequence to the eIF2alpha kinases. Working in collaboration with scientists in New York, we demonstrated that the Plasmodium kinase PfPK4 is an eIF2alpha kinase. We first fused the PfPK4 kinase domain to the constitutive dimer GST, forming GST-PfPK4-KD. GST-PfPK4-KD phosphorylated yeast eIF2alpha on Ser51 in vivo leading to inhibition of yeast cell growth. This toxicity was suppressed in cells expressing a non-phosphorylatable form of eIF2alpha in which Ser51 was replaced by Ala. In addition, our collaborators showed that PfPK4 and eIF2alpha phosphorylation are essential for the blood stage growth of Plasmodium. Thus, PfPK4 is an attractive candidate for drugs to alleviate disease and inhibit malaria transmission. In order to subvert the anti-viral defense mediated by PKR, viruses produce inhibitors of the kinase. Several members of the poxvirus family express two different types of PKR inhibitors: a pseudosubstrate inhibitor (such as the vaccinia virus K3L protein that resembles the N-terminal third of eIF2alpha) and a double-stranded RNA binding protein called E3L. High-level expression of human PKR inhibited the growth of yeast, and co-expression of the vaccinia virus K3L or E3L protein or the related variola (smallpox) virus C3L or E3L protein, respectively restored yeast cell growth. We are currently characterizing mutations in both the PKR kinase domain and its regulatory domain that confer resistance to E3L inhibition. As the kinase domain mutations enhance PKR dimerization, these results suggest that E3L may inhibit PKR by blocking kinase dimerization. We are also conducting structure-function studies on the E3L protein. Our preliminary data indicate that both the N-terminal Z-DNA binding domain (ZBD) and the C-terminal double-stranded RNA binding domain are required for PKR inhibition. However, mutations designed to disrupt nucleic acid binding to the ZBD do not affect PKR inhibition, suggesting that the ZBD has additional functions beyond nucleic acid binding. We are also studying the translation factor eIF5A. eIF5A is the sole protein containing the unusual amino acid hypusine N-epsilon-(4-amino-2-hydroxybutyl)lysine, and eIF5A was originally identified based on its ability to stimulate the yield (endpoint) of methionyl-puromycin synthesis, a model assay for first peptide bond synthesis. However, the precise cellular role of eIF5A is unknown. Using molecular genetic and biochemical studies, we recently showed that eIF5A promotes translation elongation, and this activity is dependent on the hypusine modification. As eIF5A is a structural homolog of the bacterial protein EF-P, we propose that eIF5A/EF-P is a universally conserved translation elongation factor. We are currently identifying and characterizing mutants of eIF5A.